Metabolic biomarkers for memory loss

ABSTRACT

The present invention relates to methods of determining if a subject has an increased risk of suffering from memory impairment. The methods comprise analyzing at least one plasma sample from the subject to determine a value of the subject&#39;s metabolite profile and comparing the value of the subject&#39;s metabolite profile with the value of a normal metabolite profile. A change in the value of the subject&#39;s metabolite profile, over normal values is indicative that the subject has an increased risk of suffering from memory impairment compared to a normal individual.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number RO1 AG030753 awarded by the National Institutes of Health and under contract number W81XWH-09-1-0107 awarded by the Department of Defense. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to methods of determining if a subject has an increased risk of suffering from memory impairment. The methods comprise analyzing at least one plasma sample from the subject to determine a value of the subject's metabolite profile and comparing the value of the subject's metabolite profile with the value of a normal metabolite profile. A change in the value of the subject's metabolite profile, over normal values is indicative that the subject has an increased risk of suffering from memory impairment compared to a normal individual.

Background of the Invention

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive dementia that insidiously and inexorably robs older adults of their memory and other cognitive abilities. The prevalence of AD is expected to double every 20 years from 35.6 million individuals worldwide in 2010 to 115 million affected individuals by 2050. There is no cure and current therapies are unable to slow the progression of the disease.

Early detection of the at-risk population (preclinical), or those in the initial symptomatic stages (prodromal, or mild cognitive impairment, MCI) of AD, may present opportunities for more successful therapeutic intervention, or even disease prevention by interdicting the neuropathological cascade that is ultimately characterized by the deposition of extracellular β-amyloid (Aβ) and accumulation of intracellular fibrils of hyperphosphorylated microtubule associated protein tau (MAPT or tau) within the brain. Biomarkers for early disease, including cerebrospinal fluid (CSF) tau and Aβ levels, structural and functional magnetic resonance imaging (MRI), and the recent use of brain positron emission tomography (PET) amyloid imaging, are of limited use as widespread screening tools since they provide diagnostic options that are either invasive (i.e., require lumbar puncture), time-consuming (i.e., several hours in a scanner for most comprehensive imaging protocols), expensive, or have been primarily validated in comparisons between normal subjects and those with the disease. No current blood-based biomarkers can detect incipient dementia with the required sensitivity and specificity during the preclinical stages. Continued interest in blood-based biomarkers remains because these specimens are obtained using minimally invasive, rapid, and relatively inexpensive methods. With recent technological advances in ‘omics’ technologies and systems biology analytic approaches, the comprehensive bioinformatic analyses of blood-based biomarkers may not only yield improved accuracy in predicting those at risk, but may also provide new insights into the underlying mechanisms and pathobiological networks involved in AD and possibly herald the development of new therapeutic strategies.

The preclinical interval resulting in MCI or AD is known to be variable, multifactorial, and extends for at least 7-10 years prior to the emergence of clinical signs (prodromal or manifest disease). In the absence of accurate and easily obtained biomarkers, multimodal neurocognitive testing remains the most accurate, standardized, and widely used pre-mortem screening method to determine the presence or absence of clinical MCI or AD. The utility of strict cognitive assessment for preclinical stages of MCI or AD is limited, however, as this approach is not only time-consuming but is expected, by definition, to be normal in preclinical subjects. Neuropsychological testing is able to quantitatively delineate specific brain alterations from normal, such as memory, attention, language, visuoperceptual, and executive functions, which are typically not affected in individuals during the preclinical stages. Thus, information obtained from multiple diagnostic studies will probably be most useful in defining the MCI/AD preclinical stages, including neuropsychological testing and some form(s) of biomarker(s). While CSF and neuroimaging have been used to define preclinical MCI/AD to date, their clinical utility as screening tools for asymptomatic individuals is not established.

SUMMARY OF THE INVENTION

The present invention relates to methods of determining if a subject has an increased risk of suffering from memory impairment. The methods comprise analyzing at least one plasma sample from the subject to determine a value of the subject's metabolite profile and comparing the value of the subject's metabolite profile with the value of a normal metabolite profile. A change in the value of the subject's metabolite profile, over normal values is indicative that the subject has an increased risk of suffering from memory impairment compared to a normal individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts box and whisker plots of the combined discovery and validation samples on the five composite cognitive Z-score measures.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of determining if a subject has an increased risk of suffering from memory impairment. The methods comprise analyzing at least one plasma sample from the subject to determine a value of the subject's metabolite profile and comparing the value of the subject's metabolite profile with the value of a normal metabolite profile. A change in the value of the subject's metabolite profile, over or under normal values is indicative that the subject has an increased risk of suffering from memory impairment compared to a normal individual.

As used herein, the term subject or “test subject” indicates a mammal, in particular a human or non-human primate. The test subject may or may not be in need of an assessment of a predisposition to memory impairment. For example, the test subject may have a condition or may have been exposed to injuries or conditions that are associated with memory impairment prior to applying the methods of the present invention. In another embodiment, the test subject has not been identified as a subject that may have a condition or may have been exposed to injuries or conditions that are associated with memory impairment prior to applying the methods of the present invention.

As used herein, the phrase “memory impairment” means a measureable or perceivable decline or decrease in the subject's ability to recall past events. As used herein, the term “past events” includes both recent (new) events (short-term memory) or events further back in time (long-term memory). In one embodiment, the methods are used to assess an increased risk of short-term memory impairment. In another embodiment, the methods are used to assess an increased risk in long-term memory impairment. The memory impairment can be age-related memory impairment. The memory impairment may also be disease-related memory impairment. Examples of disease-related memory impairment include but are not limited to Alzheimer's Disease, Parkinson's Disease, Multiple Sclerosis, Huntington's Disease, Pick's Disease, Progressive Supranuclear Palsy, Brain Tumor(s), Head Trauma, and Lyme Disease to name a few. In one embodiment, the memory impairment is related to amnestic mild cognitive impairment (aMCI). In another embodiment, the memory impairment is related to Alzheimer's Disease. The root cause of the memory impairment is not necessarily critical to the methods of the present invention. The measureable or perceivable decline in the subject's ability to recall past events may be assessed clinically by a health care provider, such as a physician, physician's assistant, nurse, nurse practitioner, psychologist, psychiatrist, hospice provider, or any other provider that can assess a subject's memory. The measureable or perceivable decline in the subject's ability to recall past events may be assessed in a less formal, non-clinical manner, including but not limited to the subject himself or herself, acquaintances of the subject, employers of the subject and the like. The invention is not limited to a specific manner in which the subject's ability to recall past events is assessed. In fact, the methods of the invention can be implemented without the need to assess a subject's ability to recall past events. Of course, the methods of the present invention may also include assessing the subject's ability to assess past events one or more times, both before determining the subject's metabolite profile after determining the subject's metabolite profile at least one time.

In one embodiment, the decline or decrease in the ability to recall past events is relative to each individual's ability to recall past events prior to the diagnosed decrease or decline in the ability to recall past events. In another embodiment, the decline or decrease in the ability to recall past events is relative to a population's (general, specific or stratified) ability to recall past events prior to the diagnosed decrease or decline in the ability to recall past events.

As used herein, the term means “increased risk” is used to mean that the test subject has an increased chance of developing or acquiring memory impairment compared to a normal individual. The increased risk may be relative or absolute and may be expressed qualitatively or quantitatively. For example, an increased risk may be expressed as simply determining the subject's metabolite profile and placing the patient in an “increased risk” category, based upon previous population studies. Alternatively, a numerical expression of the subject's increased risk may be determined based upon the metabolite profile. As used herein, examples of expressions of an increased risk include but are not limited to, odds, probability, odds ratio, p-values, attributable risk, relative frequency, positive predictive value, negative predictive value, and relative risk.

For example, the correlation between a subject's metabolite profile and the likelihood of suffering from memory impairment may be measured by an odds ratio (OR) and by the relative risk (RR). If P(R⁺) is the probability of developing memory impairment for individuals with the risk profile (R) and P(R⁻) is the probability of developing memory impairment for individuals without the risk profile, then the relative risk is the ratio of the two probabilities: RR=P(R⁺)/P(R⁻).

In case-control studies, however, direct measures of the relative risk often cannot be obtained because of sampling design. The odds ratio allows for an approximation of the relative risk for low-incidence diseases and can be calculated: OR=(F⁺/(1−F⁺))/(F⁻/(1−F⁻)), where F⁺ is the frequency of a risk profile in cases studies and F⁻ is the frequency of risk profile in controls. F⁺ and F⁻ can be calculated using the metabolite profile frequencies of the study.

The attributable risk (AR) can also be used to express an increased risk. The AR describes the proportion of individuals in a population exhibiting memory impairment due to a specific member of the metabolite risk profile. AR may also be important in quantifying the role of individual components (specific member) in disease etiology and in terms of the public health impact of the individual marker. The public health relevance of the AR measurement lies in estimating the proportion of cases of memory impairment in the population that could be prevented if the profile or individual component were absent. AR may be determined as follows: AR=P_(E)(RR−1)/(P_(E)(RR−1)+1), where AR is the risk attributable to a profile or individual component of the profile, and P_(E) is the frequency of exposure to a profile or individual component of the profile within the population at large. RR is the relative risk, which can be approximated with the odds ratio when the profile or individual component of the profile under study has a relatively low incidence in the general population.

In one embodiment, the increased risk of a patient can be determined from p-values that are derived from association studies. Specifically, associations with specific profiles can be performed using regression analysis by regressing the metabolite profile with memory impairment. In addition, the regression may or may not be corrected or adjusted for one or more factors. The factors for which the analyses may be adjusted include, but are not limited to age, sex, weight, ethnicity, geographic location, fasting state, state of pregnancy or post-pregnancy, menstrual cycle, general health of the subject, alcohol or drug consumption, caffeine or nicotine intake and circadian rhythms, and the subject's apolipoprotein E (ApoE) genotype to name a few.

Increased risk can also be determined from p-values that are derived using logistic regression. Binomial (or binary) logistic regression is a form of regression which is used when the dependent is a dichotomy and the independents are of any type. Logistic regression can be used to predict a dependent variable on the basis of continuous and/or categorical independents and to determine the percent of variance in the dependent variable explained by the independents; to rank the relative importance of independents; to assess interaction effects; and to understand the impact of covariate control variables. Logistic regression applies maximum likelihood estimation after transforming the dependent into a “logit” variable (the natural log of the odds of the dependent occurring or not). In this way, logistic regression estimates the probability of a certain event occurring. These analyses are conducted with the program SAS.

SAS (“statistical analysis software”) is a general purpose package (similar to Stata and SPSS) created by Jim Goodnight and N.C. State University colleagues. Ready-to-use procedures handle a wide range of statistical analyses, including but not limited to, analysis of variance, regression, categorical data analysis, multivariate analysis, survival analysis, psychometric analysis, cluster analysis, and nonparametric analysis.

As used herein, the phrase “metabolite profile” means the combination of a subject's metabolites found in the peripheral blood or portions thereof, such as but not limited to plasma or serum. The metabolite profile is a collection of measurements, such as but not limited to a quantity or concentration, for individual metabolites taken from a test sample of the subject. Examples of test samples or sources of components for the metabolite profile include, but are not limited to, biological fluids, which can be tested by the methods of the present invention described herein, and include but are not limited to whole blood, such as but not limited to peripheral blood, serum, plasma, cerebrospinal fluid, urine, amniotic fluid, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like. Test samples to be assayed also include but are not limited to tissue specimens including normal and abnormal tissue.

Techniques to assay levels of individual components of the metabolite profile from test samples are well known to the skilled technician, and the invention is not limited by the means by which the components are assessed. In one embodiment, levels of the individual lipid components of the metabolite profile are assessed using mass spectrometry in conjunction with ultra-performance liquid chromatography (UPLC), high-performance liquid chromatography (HPLC), and UPLC to name a few. Other methods of assessing levels of the individual components include biological methods, such as but not limited to ELISA assays.

The assessment of the levels of the individual components of the metabolite profile can be expressed as absolute or relative values and may or may not be expressed in relation to another component, a standard an internal standard or another molecule of compound known to be in the sample. If the levels are assessed as relative to a standard or internal standard, the standard may be added to the test sample prior to, during or after sample processing.

To assess levels of the individual components of the metabolite profile, a sample is taken from the subject. The sample may or may not processed prior assaying levels of the components of the metabolite profile. For example, whole blood may be taken from an individual and the blood sample may be processed, e.g., centrifuged, to isolate plasma or serum from the blood. The sample may or may not be stored, e.g., frozen, prior to processing or analysis.

Individual components of the metabolite profile include but are not limited to C3, PC aa C32:0, PC aa C40:5, C5-OH (C3-DC-M), PC ae C34:0, C18:1-OH, PC aa C34:4, PC aa C38:4, C5, PC aa C38:3, lysoPC a C18:0, C10:2, C12:1, C16:2, C10:1, asparagine (Asn) and asymmetric dimethylarginine (ADMA). The components are well known in the art. Briefly, compound C species, e.g., C3, denote acylcarnitines (ACs). Phosphocholine (PC) and C metabolites display combined numbers of carbon atoms for their two acyl groups (sn1 and sn2 positions), e.g., C34, whereas the combined number of double bonds (unsaturation) is displayed after the colon, e.g., C34:4. Acyl group linkages to choline backbone for PCs feature ester (a) or ether (e) linkage, e.g., PC aa C34:4. In one embodiment, the individual levels of each of the metabolites are lower than those compared to normal levels. In another embodiment, one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16 or 17 of the levels of each of the metabolites are lower than normal levels while others are higher than normal levels.

The levels of depletion or augmentation of the metabolites compared to normal levels can vary. In one embodiment, the levels of any one or more of the metabolites is at least 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 lower than normal levels. For the purposes of the present invention, the number of “times” the levels of a metabolite is lower or higher over normal can be a relative or absolute number of times. In the alternative, the levels of the metabolites may be normalized to a standard and these normalized levels can then be compared to one another to determine if a metabolite is lower or higher.

For the purposes of the present invention the metabolite profile comprises at least two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16 or all 17 of the metabolites listed above. If two metabolites are used in generating the metabolite profile, any combination of the two listed above can be used. If three metabolites are used in generating the metabolite profile, any combination of three of the metabolites listed above can be used. If four metabolites are used in generating the metabolite profile, any combination of four of the metabolites listed above can be used. If five metabolites are used in generating the metabolite profile, any combination of five of the metabolites listed above can be used. If six metabolites are used in generating the metabolite profile, any combination of six of the metabolites listed above can be used. If seven metabolites are used in generating the metabolite profile, any combination of seven of the metabolites listed above can be used. If eight metabolites are used in generating the metabolite profile, any combination of eight of the metabolites listed above can be used. If nine metabolites are used in generating the metabolite profile, any combination of nine of the metabolites listed above can be used. If ten metabolites are used in generating the metabolite profile, any combination of ten of the metabolites listed above can be used. If 11 metabolites are used in generating the metabolite profile, any combination of 11 of the metabolites listed above can be used. If 12 metabolites are used in generating the metabolite profile, any combination of 12 of the metabolites listed above can be used. If 13 metabolites are used in generating the metabolite profile, any combination of 13 of the metabolites listed above can be used. If 14 metabolites are used in generating the metabolite profile, any combination of 14 of the metabolites listed above can be used. If 15 three metabolites are used in generating the metabolite profile, any combination of 15 of the metabolites listed above can be used. If 16 metabolites are used in generating the metabolite profile, any combination of 16 of the metabolites listed above can be used. Of course, all 17 metabolites can be are used in generating the metabolite profile.

Table 1 below lists an exemplary analysis of the metabolites. The normalized ratios depict relative abundance of metabolites in the C_(pre) group (noted here as Con-pre) as compared to the NC group. Herein, a ratio of one (1) indicates no change while values less than one indicate decreased abundance in the diagnostic group as compared to the NC, or vice versa.

TABLE 1 Metabolite Levels Discovery Set Validation Set log log log log Metabolite ratio ratio ratio ratio Name p value (mean) (median) (mean) (median) C3 0.009020331 −0.45 −0.27 −1.28 −0.33 PC aa C32:0 0.015037221 −0.40 −0.18 −1.46 −0.63 PC aa C40:5 0.029816569 −0.43 −0.15 −0.59 −0.42 C5-OH 0.000335083 −0.42 −0.32 −2.12 −2.02 (C3-DC-M) PC æ C34:0 0.01482628 −0.41 −0.23 −1.27 −0.44 C18:1-OH 0.018167307 −0.27 −0.33 −1.25 −0.24 PC aa C34:4 0.034589192 −0.44 −0.19 −0.14 −0.08 Asn 0.044051559 −0.18 −0.09 −0.90 −0.19 PC aa C38:4 0.00260766 −0.57 −0.33 −0.22 −0.15 ADMA 0.016259751 −0.30 −0.11 −1.08 −1.58 C5 0.001348657 −0.28 −0.20 −0.68 −0.25 PC aa C38:3 0.043163996 −0.41 −0.15 −0.81 −0.24 lysoPC a C18:0 0.028887542 −0.46 −0.22 −0.51 −0.23 C10:2 0.040290286 −0.40 −0.19 −0.19 −0.29 C12:1 6.83E−05 0.51 0.34 0.87 0.21 C16:2 0.010847154 0.28 0.08 0.50 0.04 C10:1 0.002568803 0.36 0.22 0.61 0.02

Techniques to assay levels of individual components of any non-lipid component of the metabolite profile from test samples are well known to the skilled technician, and the invention is not limited by the means by which the components are assessed. In one embodiment, levels of the individual components of the non-lipid portion of the profile are assessed using quantitative arrays, PCR, Northern Blot analysis, Western Blot analysis, mass spectroscopy, high-performance liquid chromatography (HPLC) and the like. Other methods of assessing levels of the individual components include biological methods, such as but not limited to ELISA assays. To determine levels of metabolites, it is not necessary that an entire metabolite, e.g., a full length protein or an entire RNA transcript, be present or fully sequenced. In other words, determining levels of, for example, a fragment of protein being analyzed may be sufficient to conclude or assess that an individual component of the metabolite profile, including the lipid and non-lipid portions of the metabolite profile, being analyzed is up- or down-regulated. Similarly, if, for example, arrays or blots are used to determine metabolite levels, the presence/absence/strength of a detectable signal may be sufficient to assess levels of metabolite.

The subject's metabolite profile is compared to the profile that is deemed to be a normal metabolite profile. To establish the metabolite profile of a normal individual, an individual or group of individuals may be first assessed for their ability to recall past events to establish that the individual or group of individuals has a normal or acceptable ability memory. Once established, the metabolite profile of the individual or group of individuals can then be determined to establish a “normal metabolite profile.” In one embodiment, a normal metabolite profile can be ascertained from the same subject when the subject is deemed to possess normal cognitive abilities and no signs (clinical or otherwise) of memory impairment. In one embodiment, a “normal” metabolite profile is assessed in the same subject from whom the sample is taken prior to the onset of measureable, perceivable or diagnosed memory impairment. That is, the term “normal” with respect to a metabolite profile can be used to mean the subject's baseline metabolite profile prior to the onset of memory impairment. The metabolite profile can then be reassessed periodically and compared to the subject's baseline metabolite profile. Thus, the present invention also include methods of monitoring the progression of memory impairment in a subject, with the methods comprising determining the subject's metabolite profile more than once over a period of time. For example, some embodiments of the methods of the present invention will comprise determining the subject's metabolite profile two, three, four, five, six, seven, eight, nine, 10 or even more times over a period of time, such as a year, two years, three, years, four years, five years, six years, seven years, eight years, nine years or even 10 years or longer. The methods of monitoring a subject's risk of having memory impairment would also include embodiments in which the subject's metabolite profile is assessed during and after treatment of memory impairment. In other words, the present invention also includes methods of monitoring the efficacy of treatment of memory impairment by assessing the subject's metabolite profile over the course of the treatment and after the treatment. The treatment may be any treatment designed to increase a subject's ability to recall past events, i.e., improve a subject's memory.

In another embodiment, a normal metabolite profile is assessed in a sample from a different subject or patient (from the subject being analyzed) and this different subject does not have or is not suspected of having memory impairment. In still another embodiment, the normal metabolite profile is assessed in a population of healthy individuals, the constituents of which display no memory impairment. Thus, the subject's metabolite profile can be compared to a normal metabolite profile generated from a single normal sample or a metabolite profile generated from more than one normal sample.

Of course, measurements of the individual components, e.g., concentration, of the normal metabolite profile can fall within a range of values, and values that do not fall within this “normal range” are said to be outside the normal range. These measurements may or may not be converted to a value, number, factor or score as compared to measurements in the “normal range.” For example, a measurement for a specific metabolite that is below the normal range, may be assigned a value or −1, −2, −3, etc., depending on the scoring system devised.

In one embodiment, the “metabolite profile value” can be a single value, number, factor or score given as an overall collective value to the individual molecular components of the profile, or to the categorical components, i.e., a lipid portion and an amino acid portion. For example, if each component is assigned a value, such as above, the metabolite value may simply be the overall score of each individual or categorical value. For example, if 10 of the components of the metabolite profile are lipids, and five of the components are assigned values of “−2” and five are assigned values of “−1,” the lipid portion of the metabolite profile in this example would be −15, with a normal value being, for example, “0.” In this manner, the metabolite profile value could be useful single number or score, the actual value or magnitude of which could be an indication of the actual risk of memory impairment, e.g., the “more negative” the value, the greater the risk of memory impairment.

In another embodiment the “metabolite profile value” can be a series of values, numbers, factors or scores given to the individual components of the overall profile. In another embodiment, the “metabolite profile value” may be a combination of values, numbers, factors or scores given to individual components of the profile as well as values, numbers, factors or scores collectively given to a group of components. In another example, the metabolite profile value may comprise or consist of individual values, number, factors or scores for specific component as well as values, numbers, factors or scores for a group on components.

In another embodiment individual values from the metabolites can be used to develop a single score, such as a “combined metabolite index,” which may utilize weighted scores from the individual component values reduced to a diagnostic number value. The combined metabolite index may also be generated using non-weighted scores from the individual component values. When the “combined metabolite index” exceeds (or is less than) a specific threshold level, the individual has a high risk of memory impairment, whereas the maintaining a normal range value of the “combined metabolite index” would indicate a low or minimal risk of memory impairment. In this embodiment, the threshold value would be set by the combined metabolite index from normal subjects.

In another embodiment, the value of the metabolite profile can be the collection of data from the individual measurements and need not be converted to a scoring system, such that the “metabolite profile value” is a collection of the individual measurements of the individual components of the profile.

In specific embodiments, a subject is diagnosed of having an increased risk of suffering from memory impairment if the subject's 17, 16, 15, 14, 13, 12, 11, 10, nine, eight, seven, six, five, four, three, two or even one of the metabolites herein are at abnormal levels.

If it is determined that a subject has an increased risk of memory impairment, the attending health care provider may subsequently prescribe or institute a treatment program. In this manner, the present invention also provides for methods of screening individuals as candidates for treatment of memory impairment. The attending healthcare worker may begin treatment, based on the subject's metabolite profile, before there are perceivable, noticeable or measurable signs of memory impairment in the individual.

Similarly, the invention provides methods of monitoring the effectiveness of a treatment for memory impairment. Once a treatment regimen has been established, with or without the use of the methods of the present invention to assist in a diagnosis of memory impairment, the methods of monitoring a subject's metabolite profile over time can be used to assess the effectiveness of a memory impairment treatment. Specifically, the subject's metabolite profile can be assessed over time, including before, during and after treatments for memory impairment. The metabolite profile can be monitored, with, for example, a decline in the values of the profile over time being indicative that the treatment may not be as effective as desired.

All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications cited herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated as having been incorporated by reference in its entirety

EXAMPLES Example 1

Neurocognitive Methods

A total of 525 volunteers participated in this study as part of the Rochester/Orange County Aging Study (R/OCAS), an ongoing natural history study of cognition in community-dwelling older adults. Briefly, participants were followed with yearly cognitive assessments and blood samples were collected following an overnight fast and withholding of all medications. At baseline and each yearly visit, participants completed assessments in such as activities in daily living, memory complaints, signs and symptoms of depression, and were administered a detailed cognitive assessment.

For this study, data from the cognitive tests were used to classify participants into groups for biomarker discovery. Standardized scores (Z-scores) were derived for each participant on each cognitive test and the composite Z-scores were computed for five cognitive domains (attention, executive, language, memory, visuoperceptual) (Table 2).

TABLE 2 Visuoperceptual Attention (Z_(att)) Executive (Z_(exe)) Language (Z_(lan)) (Z_(vis)) Memory (Z_(mem)) Wechsler Wechsler 1-min Category Hooper Visual Rey Auditory Memory Scale-III Memory Scale-III fluency Organization Test Verbal Learning Forward Digit Backward Digit (Animals) (HVOT) Test Learning Span (WMS-III Span (WMS-III (RAVLT Learning) FDS) BDS) Trail Making Trail Making Boston Naming Rey Auditory Test-Part A Test-Part B Test 60-Item Verbal Learning (TMT-A) (TMT-B) version (BNT-60) Test Retrieval (RAVLT Retrieval) Rey Auditory Verbal Learning Test Retention (RAVLT Recognition)

Normative data for Z-score calculations were derived from the performance of the participants on each of the cognitive tests adjusted for age, education, sex, and visit. To reduce the effect of cognitively impaired participants on the mean and SD, age-, education-, sex, and visit-adjusted residuals from each domain Z-score model were robustly standardized to have median 0 and robust SD=1, where the robust SD=IQR/1.35, as 1.35 is the IQR (Inter-Quartile Range) of a standard normal distribution.

The participants were then categorized into groups of incident aMCI or early AD (combined into one category aMCI/AD), cognitively normal control (NC), and those who converted to MCI or AD over the course of the study (Converters) based on these composite scores. Impairment was defined as a Z-score 1.35 SD below the cohort median. All participants classified as aMCI met recently revised criteria for the amnestic subtype of MCI. Other behavioral phenotypes of MCI were excluded to concentrate on the amnestic form, which most likely represents nascent Alzheimer's pathology. All early AD participants met recently revised criteria for probable Alzheimer's disease with impairment in memory and at least one other cognitive domain. For the MCI and early AD groups, scores on the measures of memory complaints (MMQ) and activities of daily living (PGC-IADL) were used to corroborate research definitions of these states. All Converters had non-impaired memory at entry to the study (Z_(mem)≥−1.35), developed memory impairment over the course of the study (Z_(mem)≤−1.35) and met criteria for the above definitions of aMCI or AD. To enhance the specificity of the biomarker analyses, NC participants in this study were conservatively defined with Z_(mem)±1 SD of the cohort median rather than simply ≥−1.35, and all other Z-scores ≥−1.35 SD.

For each subject, Z_(mem)(last), Z_(att)(last), Z_(exe)(last), Z_(lan)(last), and Z_(vis)(last) were defined as the age-gender-education-visit-adjusted robust Z-scores for the last available visit for each subject. The aMCI/AD group was defined as those participants whose adjusted Z_(mem) was 1 IQR below the median at their last available visit, i.e., Z_(mem)(last)≤−1.35. Converters were defined as that subset of the a MCI/AD group whose adjusted Z_(mem) at baseline visit 0 was no more than 1 IQR below the median, i.e., Z_(mem)(visit=0)>−1.35 and Z_(mem)(last)≤−1.35. Participants were classified as NC if they had central scores on all domains at both the first and last visits, i.e., only if they met all of the following six conditions: (i) −1<Z_(mem)(last)<1, (ii) −1<Z_(mem)(visit=0)<1, (iii) Z_(min)(last)>−1.35, (iv) Z_(min)(visit=0)>−1.35, (v) Z_(max)(last)<1.35, and (vi) Z_(max)(visit=0)<1.35, where Z_(max)(last) and Z_(max)(visit=0) denote the maximum of the five adjusted Z-scores at the last and first visits, respectively. Z_(mem) for normal participants had to be within 0.74 IQR (1 SD) of the median, rather than just 1 IQR (1.35 SD), to guarantee that they were >0.25 IQR (0.35 SD) from a MCI/AD participants.

After three years of being in the study, (December, 2010), 202 participants had completed a baseline and two yearly visits. At the third visit, 53 participants met criteria for aMCI/AD and 96 met criteria for NC. Of the 53 aMCI/AD participants, 18 were Converters and 35 were incident aMCI or AD. The remaining 53 participants did not meet the criteria for either group and were not considered for biomarker profiling. Some of these individuals met criteria for non-amnestic MCI and many had borderline or even above average memory scores that precluded their inclusion as either aMCI/AD or NC. 53 of the NC participants were matched to the 53 aMCI/AD participants based on sex, age, and education level. Blood samples were obtained on the last available study visit for the 53 MCI/AD and the 53 NC for biomarker discovery. Two blood samples from each of the 18 Converters were also included: one from the baseline visit (C_(pre)) when Z_(mem) was non-impaired and one from the third visit (C_(post)) when Z_(mem) was impaired and they met criteria for either aMCI or AD. Thus, at total of 124 samples from 106 participants were analyzed.

Internal cross-validation was employed to validate findings from the discovery phase. Blood samples for validation were identified at the end of the fifth year of the study and all 106 participants included in the discovery phase were excluded from consideration for the validation phase. Cognitive composite Z-scores were re-calculated based on the entire sample available and the same procedure and criteria were used to identify samples for the validation phase. A total of 145 participants met criteria for a group: 21aMCI/AD and 124 NC. Of the 21 aMCI/AD, 10 were Converters. 20 of the NC participants were matched to the aMCI/AD participants on the basis of age, sex, and education level as in the discovery phase. In total, 41 participants contributed samples to the validation phase and, as before, the 10 Converters also contributed a baseline sample (C_(pre)) for a total of 51 samples.

Neurocognitive Statistical Analyses

The neurocognitive analyses were designed to demonstrate the general equivalence of the discovery and validation samples on clinical and cognitive measures. Separate Multivariate Analysis of Variance (MANOVA's) tests were used to examine discovery/validation group performance on the composite Z-scores and on self-report measures of memory complaints, memory related functional impairment, depressive symptoms, and a global measure of cognitive function. In the first MANOVA, biomarker sample (discovery, validation) was the independent variable and MMQ, IADL, GDS, and MMSE were the dependent variables. In the second MANOVA, biomarker sample (discovery, validation) was the independent variable and the five cognitive domain Z-scores (Z_(att), Z_(exe), Z_(lan), Z_(mem), and Z_(vis)) were the dependent variables. Significance was set at alpha=0.05 and Tukey's HSD procedure was used for post-hoc comparisons. All statistical analyses were performed using SPSS (version 21).

Example 2

Reagents

LC/MS-grade acetonitrile (ACN), Isopropanol (IPA), water and methanol were purchased from Fisher Scientific (New Jersey, USA). High purity formic acid (99%) was purchased from Thermo-Scientific (Rockford, Ill.). Debrisoquine, 4-Nitrobenzoic acid (4-NBA), Pro-Asn, Glycoursodeoxycholic acid, Malic acid, were purchased from Sigma (St. Louis, Mo., USA). All lipid standards including 14:0 LPA, 17:0 Ceramide, 12:0 LPC, 18:0 Lyso PI and PC(22:6/0:0) were procured from Avanti Polar Lipids Inc. (USA).

Metabolite Extraction

Briefly, the plasma samples were thawed on ice and vortexed. For metabolite extraction, 25 μL of plasma sample was mixed with 175 μL of extraction buffer (25% acetonitrile in 40% methanol and 35% water) containing internal standards [10 μL of debrisoquine (1 mg/mL), 50 μL of 4, nitrobenzoic acid (1 mg/mL), 27.3 μl of Ceramide (1 mg/mL) and 2.5 μL of LPA (lysophosphatidic acid) (4 mg/mL) in 10 mL). The samples were incubated on ice for 10 minutes and centrifuged at 14,000 rpm at 4° C. for 20 minutes. The supernatant was transferred to a fresh tube and dried under vacuum. The dried samples were reconstituted in 200 μL of buffer containing 5% methanol, 1% acetonitrile and 94% water. The samples were centrifuged at 13,000 rpm for 20 minutes at 4° C. to remove fine particulates. The supernatant was transferred to a glass vial for UPLC-ESI-Q-TOF-MS analysis.

Each sample (2 μL) was injected onto a reverse-phase CSH C18 1.7 μM 2.1×100 mm column using an Acquity H-class UPLC system (Waters Corporation, USA). The gradient mobile phase comprised of water containing 0.1% formic acid solution (Solvent A), 100% acetonitrile (Solvent B) and 10% acetonitrile in isopropanol (IPA) containing 0.1% formic acid and 10 mM ammonium formate (Solvent C). Each sample was resolved for 13 minutes at a flow rate of 0.5 mL/min for 8 min and then 0.4 mL/min from 8 to 13 min. The UPLC gradient consisted of 98% A and 2% B for 0.5 min then a ramp of curve 6 to 60% B and 40% A from 0.5 min to 4.0 min, followed by a ramp of curve 6 to 98% B and 2% A from 4.0 to 8.0 min, then ramped to 5% B and 95% C from 9.0 min to 10.0 min at a flow rate of 0.4 ml/min, and finally to 98% A and 2% B from 11.0 min to 13 minutes. The column eluent was introduced directly into the mass spectrometer by electrospray ionization. Mass spectrometry was performed on a Quadrupole-Time of Flight (Q-TOF) instrument (Xevo G2 QTOF, Waters Corporation, USA) operating in either negative (ESI⁻) or positive (ESI⁺) electrospray ionization mode with a capillary voltage of 3200 V in positive mode and 2800 V in negative mode, and a sampling cone voltage of 30 V in both modes. The desolvation gas flow was set to 750 L h⁻¹ and the temperature was set to 350° C. while the source temperature was set at 120° C. Accurate mass was maintained by introduction of a lock spray interface of leucine-enkephalin (556.2771 [M+H]⁺ or 554.2615 [M−H]⁻) at a concentration of 2 pg/μl in 50% aqueous acetonitrile and a rate of 2 μl/min. Data were acquired in centroid MS mode from 50 to 1200 m/z mass range for TOF-MS scanning as single injection per sample and the batch acquisition was repeated to check experimental reproducibility. For the metabolomics profiling experiments, pooled quality control (QC) samples (generated by taking an equal aliquot of all the samples included in the experiment) were run at the beginning of the sample queue for column conditioning and every ten injections thereafter to assess inconsistencies that are particularly evident in large batch acquisitions in terms of retention time drifts and variation in ion intensity over time. This approach has been recommended and used as a standard practice by leading metabolomics researchers. A test mix of standard metabolites was run at the beginning and at the end of the run to evaluate instrument performance with respect to sensitivity and mass accuracy. The sample queue was randomized to remove bias.

Stable Isotope Dilution—Multiple Reaction Monitoring Mass Spectrometry (SID-MRM-MS)

Targeted metabolomic analysis of plasma sample was performed using the Biocrates Absolute-IDQ P180 (BIOCRATES, Life Science AG, Innsbruck, Austria). This validated targeted assay allows for simultaneous detection and quantification of metabolites in plasma samples (10 μL) in a high throughput manner. The methods have been described in detail. The plasma samples were processed as per the instructions by the manufacturer and analyzed on a triple quadrupole mass spectrometer (Xevo TQ-S, Waters Corporation, USA) operating in the MRM mode. The measurements were made in a 96 well format for a total of 148 samples, seven calibration standards and three quality control samples were integrated in the kit.

Briefly, the flow injection analysis (FIA) tandem mass spectrometry (MS/MS) method was used to quantify a panel of 144 lipids simultaneously by multiple reaction monitoring. The other metabolites were resolved on the UPLC and quantified using scheduled MRMs. The kit facilitated absolute quantitation of 21 amino acids, hexose, carnitine, 39 acylcarnitines, 15 sphingomyelins, 90 phosphatidylcholines and 19 biogenic amines. Data pre-processing was performed using the MetIQ software (Biocrates) while the statistical analyses were performed using linear regression as well as the STAT pack module v3 (Biocrates). The concentration is expressed as nmol/L. Quality control samples were used to assess reproducibility of the assay. The mean of the coefficient of variation (CV) for the 180 metabolites was 0.08 and 95% of the metabolites had a CV of <0.15.

Metabolite Statistical Analyses

The m/z features of metabolites were normalized with log transformation that stabilized the variance followed with a quantile normalization to make the empirical distribution of intensities the same across samples. The metabolites were selected among all those known to be identifiable using a ROC regularized learning technique, based on the least absolute shrinkage and selection operator (LASSO) penalty as implemented with the R package ‘glmnet’, which uses cyclical coordinate descent in a pathwise fashion. The regularization path over a grid of values was obtained for the tuning parameter lambda through 10-fold cross-validation. The optimal value of the tuning parameter lambda, which was obtained by the cross-validation procedure, was then used to fit the model. All the features with non-zero coefficients were retained for subsequent analysis. The classification performance of the selected metabolites was assessed using area under the ROC (receiver operating characteristic) curve (AUC). The ROC can be understood as a plot of the probability of classifying correctly the positive samples against the rate of incorrectly classifying true negative samples. Thus the AUC measure of an ROC plot is actually a measure of predictive accuracy.

Results

Over the course of the study, 74 participants met criteria for either aMCI or mild AD, 46 of these were incidental cases at entry and 28 phenoconverted (Converters) from a non-impaired memory status at entry (C_(pre)). The average time to phenoconversion was 2.1 years (range=1-5 years). 53 aMCI/AD participants were selected, including 18 Converters, and 53 age-, education-, and sex-matched cognitively normal control (NC) participants for untargeted lipidomics biomarker discovery. Internal cross validation was used to evaluate the accuracy of the discovered metabolite profile in classifying a blinded sample of 51 subjects consisting of the remaining subset of 21 aMCI/AD participants, including 10 Converters, and an additional 20 matched NC.

The aMCI/AD, Converter, and NC groups were defined primarily using a composite measure of memory performance in addition to composite measures of other cognitive abilities and clinical measures of memory complaints and functional capacities. (See Tables 2 and 3).

TABLE 3 Dependent Measure Domain Clinical/Cognitive Measures (Range) Assessed Multiple Assessment Inventory IADL Scale (MAI-IADL) Total Score Functional Lawton M P. (1988) Instrumental Activities of Daily Living (0-27) capacities (IADL) scale: Original observer-rated version. Psychopharmacology Bulletin, 24, 785-7. Multifactorial Memory Questionnaire (MMQ) Total Score Memory Troyer A K and Rich J B. (2002). Psychometric properties of a (0-228) complaints new metamemory questionnaire for older adults. Journal of Gerontology, 57(1), 19-27. Mini Mental State Examination (MMSE) Total Score Global cognitive Folstein, M F, Folstein, S E, and McHugh, P R. (1975). “Mini- (0-30) ability mental state”. Journal of Psychiatric Research, 12, 189-98. Geriatric Depression Scale-Short Form (GDS-SF) Total Score Mood Sheikh J I and Yesavage J A. (1986). Geriatric Depression (0-15) Scale (GDS): Recent evidence and development of a shorter version. Clinical Gerontologist, 5, 165-173. Wechsler Memory Scale-III Forward Digit Span Span Length Attention (WMS-III FDS) (0-9) Wechsler D. Wechsler Memory Scale-III Manual. San Antonio, TX: The Psychological Corporation, 1997. Trail Making Test-Part A (TMT-A) Completion time Attention Reitan R M. (1958). Validity of the Trail Making Test as an (1-300 seconds) indicator of organic brain damage. Perceptual and Motor Skills, 8, 271-6. Wechsler Memory Scale-III Backward Digit Span Span Length Executive ability (WMS-III BDS) (0-8) Wechsler D. Wechsler Memory Scale-III Manual. San Antonio, TX: The Psychological Corporation, 1997. Trail Making Test-Part B (TMT-B) Completion Time Executive ability Reitan R M. (1958). Validity of the Trail Making Test as an (1-300 seconds) indicator of organic brain damage. Perceptual and Motor Skills, 8, 271-6. Category fluency (Animals) Animals named in Language Borkowski J, Benton A, Spreen O. (1967). Word fluency and 1-minute brain damage. Neuropsychologia, 5, 135-140 Boston Naming Test 60-Item version (BNT-60) Total Correct Language Kaplan E, Goodglass H, and Weintraub S. (1983). Boston (0-60) Naming Test. Philadelphia: Lea & Feibiger. Rey Auditory Verbal Learning Test Learning (RAVLT Total words Verbal learning Learning) recalled over Trials Rey A. (1964). L'examen clinique en psychologie. Paris: 1-5 (0-75) Presses Universitaires de France. Rey Auditory Verbal Learning Test Recall (RAVLT Words recalled at Verbal retrieval Retrieval) 20-minute delay (0-15) Rey A. (1964). L'examen clinique en psychologie. Paris: Presses Universitaires de France. Rey Auditory Verbal Learning Test Retention (RAVLT True positives - Verbal Recognition) false positives retention Rey A. (1964). L'examen clinique en psychologie. Paris: (0-15) Presses Universitaires de France. Hooper Visual Organization Test (HVOT) Total score Visuoperception Hooper H E. Hooper Visual Organization Test (VOT) Los (0-30) Angeles: Western Psychological Services; 1983.

To confirm the reproducibility of the metabolite panel from the discovery samples, targeted quantitative metabolomics analyses was performed using plasma from the independent validation group of 40 participants as an independent cross validation. One sample from the MCI/AD group was not available for metabolomic analysis. The validation samples were obtained from the last available visit from the aMCI/AD, C_(pre), and NC groups and were designated as groups A, B, and C and analyzed in a blinded fashion, without specification of diagnostic identities. The samples were processed and analyzed using the same SID-MRM-MS technique as in the discovery phase. The blinded data were statistically analyzed to determine if the unknown groups could be characterized into the correct diagnostic categories based solely on the levels of the metabolite panel.

The metabolomic data was used from the untargeted LASSO analysis to build separate linear classifier models that would distinguish the aMCI/AD group from the NC group and the C_(pre) group from the NC group. A receiver operating characteristic (ROC) analysis was employed to determine the area under the ROC curve (AUC) to assess the performance of the classifier models in differentiating the groups. When distinguishing between the C_(pre) and NC groups, the LASSO identified metabolites yielded an AUC of 0.96 and 0.83 when distinguishing the aMCI/AD and NC groups. These high AUC values demonstrate robust discrimination between the aMCI/AD, C_(pre), and NC groups by the models. Using the same linear classifier method, a simple logistic model was constructed using just the metabolite panel and used this to classify the same groups. When displayed as a ROC curve, the ten metabolite panel classified C_(pre) and NC participants with an AUC of 0.96, while the panel yielded an AUC of 0.827 for the aMCI/AD vs NC classification. 

What is claimed is:
 1. A method of treating memory impairment in a subject, the method comprising a) analyzing at least one plasma sample from the subject in a fasting state to determine the concentrations of each component of a set of metabolites, wherein the components comprise C3, phosphocholine (PC) aa C32:0, PC aa C40:5, C5-OH (C3-DC-M), PC ae C34:0, C18:1-OH, PC aa C34:4, PC aa C38:4, C5, PC aa C38:3, lysoPC a C18:0, C10:2, C12:1, C16:2, C10:1, asparagine (Asn) and asymmetric dimethylarginine (ADMA), and b) administering a treatment for memory impairment to the subject when: (i) the concentrations of the components C3, PC aa C32:0, PC aa C40:5, C5-OH (C3-DC-M), PC ae C34:0, C18:1-OH, PC aa C34:4, PC aa C38:4, C5, PC aa C38:3, lysoPC a C18:0, C10:2, Asn, and ADMA in the subject's set of metabolites are lower as compared to their concentrations in a set of metabolites from subjects determined to define normal concentrations, and (ii) the concentrations of the components C12:1, C16:2, and C10:1 in the subject's set of metabolites are higher as compared to their concentrations in the set of metabolites from subjects determined to define normal concentrations.
 2. The method of claim 1, wherein the normal concentrations comprise concentrations of each component of the subject's set of metabolites prior to the onset of memory impairment.
 3. The method of claim 1, wherein the normal concentrations comprise concentrations of each component from a population of individuals that do not display memory impairment.
 4. The method of claim 1, wherein the concentrations of the set of metabolites from the subject are measured using mass spectroscopy.
 5. The method of claim 1, further comprising monitoring effectiveness of the treatment for memory impairment. 